COMPOSITIONS AND METHODS EMPLOYING UNIVERSAL-BINDING NUCLEOTIDES FOR TARGETING MULTIPLE GENE VARIANTS WITH A SINGLE siRNA DUPLEX

Inactive Publication Date: 2007-11-01
MDRNA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]The present disclosure fulfills these and other related needs by providing compositions and methods for increasing the number of target RNAs, such as variants of viral RNAs or endogenous genes, that are susceptible to degradation facilitated by one or more small inhibitory nucleic acid(s

Problems solved by technology

This approach, however, employs a single RNA target methodology and ignores the possibility that variants of the target RNA (gene variants) may be present or later developed within the cell.
Thus, the introduction of an siRNA with a specific nucleotide sequence may target a particular mRNA for destruction, yet remain ineffective in destroying variants of that RNA.
In this

Method used

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  • COMPOSITIONS AND METHODS EMPLOYING UNIVERSAL-BINDING NUCLEOTIDES FOR TARGETING MULTIPLE GENE VARIANTS WITH A SINGLE siRNA DUPLEX
  • COMPOSITIONS AND METHODS EMPLOYING UNIVERSAL-BINDING NUCLEOTIDES FOR TARGETING MULTIPLE GENE VARIANTS WITH A SINGLE siRNA DUPLEX
  • COMPOSITIONS AND METHODS EMPLOYING UNIVERSAL-BINDING NUCLEOTIDES FOR TARGETING MULTIPLE GENE VARIANTS WITH A SINGLE siRNA DUPLEX

Examples

Experimental program
Comparison scheme
Effect test

Example

Example 1

Stability of Universal-Binding Nucleotide Comprising siRNA in Rat Plasma

[0146]This Example discloses a suitable animal model system for determining the in vivo stability of a universal-binding nucleotide comprising siRNA of the present disclosure.

[0147]A 20 μg aliquot of each universal-binding nucleotide comprising siRNA duplex of are mixed with 200 μl of fresh rat plasma incubated at 37° C. At various time points (0, 30, 60 and 20 min), 50 μl of the mixture are taken out and immediately extracted by phenol:chloroform. SiRNAs are dried following precipitation by adding 2.5 volumes of isopropanol alcohol and subsequent washing step with 70% ethanol. After dissolving in water and gel loading buffer the samples are analyzed on 20% polyacrylamide gel, containing 7 M urea and visualized by ethidium bromide staining and quantitated by densitometry. The level of degradation at each time point may be assessed by electrophoresis on a PAGE gel.

Example

Example 2

Measurement of Gene Knockdown Activity by Universal-Binding Nucleotide Comprising siRNA

[0148]This Example demonstrates the utility of siRNA containing the universal-binding nucleotide ribo-inosine (I) at locations within the antisense strand that correspond to sites of sequence variation in the NP gene from a number of influenza isolates.

[0149]Each of the six variant influenza virus NP gene sequences was cloned into the psiCHECK™-2 plasmid vector (Promega, Madison, Wis.). The psiCHECK™-2 plasmid vector is designed to provide a quantitative and rapid assessment of RNA interference (RNAi) by monitoring changes in expression of a target gene (i.e., influenza virus NP gene variant CM01-06) fused to a Renilla luciferase reporter gene. The influenza NP gene was cloned into the psiCHECK™-2 plasmid within its multiple cloning region, which is downstream of the Renilla translational stop codon, thereby generating a fusion mRNA. Initiation of the RNAi process by one or more of the GM...

Example

Example 3

Measurement of Off Target Effect by Universal-Binding Nucleotide Comprising siRNA

[0156]This Example provides a suitable methodology for measuring off-target effects mediated by universal-binding nucleotide comprising siRNA of the present disclosure.

[0157]Although siRNA of the present disclosure may be suitably employed for disrupting the expression of variant target genes, there remains the possibility that such siRNA may affect the expression of one or more non-target gene(s). Thus, an off-target profile may be generated for siRNAs that target a variant of an otherwise wild-type gene, such as a viral gene or an endogenous gene. Agilent microarrays may be employed that consist of 60-mer probe oligonucleotide targets representing, for example, 18,500 well-characterized, full-length human genes.

[0158]It is expected that siRNA modifications will have a significant effect on reducing off-target responses. The extent of G:U nucleotide pairing in all the identified siRNA off-targ...

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Abstract

Provided are siRNA molecules of between about 15 base-pairs and about 40 base-pairs comprising one or more universal-binding nucleotide such as inosine, 1-β-D-ribofuranosyl-5-nitroindole, and 1-β-D-ribofuranosyl-3-nitropyrrole, compositions comprising one or more universal-binding nucleotide comprising siRNA, and methods for making and for using such universal-binding nucleotide comprising siRNA molecules to increase the specific binding of the modified siRNA molecule to variants of a target sequence such as, for example, when in contact with a biological sample and to reduce off-target effects of the siRNA molecule.

Description

[0001]This patent application claims priority under 35 U.S. § 119(e) of U.S. Provisional Application No. 60 / 796,274 filed Apr. 27, 2006, the contents of which are incorporated herein by reference.BACKGROUND OF THE DISCLOSURE[0002]1. Technical Field of the Invention[0003]The present invention relates to the treatment of disorders by means of RNA interference (RNAi). More specifically, the present disclosure relates to the targeted delivery of small inhibitory nucleic acid molecules (siRNA) that are capable of mediating RNAi against genes, and variants thereof, wherein the siRNA comprise one or more universal-binding nucleotide such as, for example, inosine, 1-β-D-ribofuranosyl-5-nitroindole, and 1-β-D-ribofuranosyl-3-nitropyrrole.[0004]2. Description of the Related Art[0005]RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by small inhibitory nucleic acid molecules (siRNAs) a double-stranded RNA (dsRNA) that is homolog...

Claims

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Application Information

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IPC IPC(8): C12N5/06C07H21/02C12N15/11C12N15/113
CPCC12N15/111C12N15/113C12N2310/14C12N2310/321C12N2310/33C12N2320/51C12N2310/331C12N2320/50C12N2310/3521A61P35/00
Inventor QUAY, STEVEN C.MCSWIGGEN, JAMES ANTHONY
Owner MDRNA
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